1. Field of the Invention
The present invention relates generally to reverse-turn mimetics, including inhibitors of cell adhesion-mediated disease, central nervous system disorders, and several other disorders as well as to a chemical library of reverse-turn mimetics.
2. Description of the Related Art
In the search for new therapeutics, the pharmaceutical industry has increasingly turned to the techniques of combinatorial chemistry, parallel synthesis, and high throughput screening to generate and optimize lead compounds (Combinatorial Chemistry and Molecular Diversity in Drug Discovery Gordon and Kerwin, Eds., John Wiley & Sons, New York, 1998; The Combinatorial Index Bunin, Academic Press, New York, 1998; A Practical Guide to Combinatorial Chemistry Czarnik and DeWitt, Eds., American Chemical Society, Washington, D.C., 1997; High Throughput Screening: The Discovery of Bioactive Substances Devlin, Marcel Dekker, N.Y., 1997). These techniques can produce libraries of hundreds to hundreds of thousands—or more—of compounds in a short period of time. The libraries are then assayed against targets of interest, often in a highly automated fashion, to identify biologically active compounds. Libraries, which are simply collections of compounds, may be tightly focused around a specific template or contain a variety of unrelated templates. In many instances, the diversity of the library is an important design parameter.
On a basic level, the number of points of diversity on a molecular template or scaffold, i.e., the number of positions at which variation in structure may be introduced, has a practical effect on the ease with which large libraries may be created. When combinatorial techniques are employed, a template that contains three points of diversity would give rise to 8000 compounds if 20 components are used to derivatize each point and a total of 60 reactions are carried out (203). However, a template with four points of diversity will yield over 50,000 compounds when 15 components are used at each point in a total of 60 reactions (154). In general, large libraries may be created more efficiently on templates allowing more possibilities for derivatization.
In order to increase the chances of finding a biologically active compound for a particular target, it is usually desirable to synthesize a library spanning a range of both conformational space and chemical properties such as hydrophobicity and hydrogen bonding ability. At the same time, low molecular weight is often a goal as well, since compounds of less than 500 Daltons are perceived as more likely to have favorable pharmacokinetic properties in relation to higher molecular weight compounds. All these characteristics point to the continuing need for small compact templates that support a wide range of substituents and which are simple to synthesize.
Reverse-turns comprise one of three classes of protein secondary structure and display three (gamma-turn), four (beta-turns), or more (loops) amino acid side chains in a fixed spatial relationship to each other. Turns have proven important in molecular recognition events (Rose et al., Advances in Protein Chemistry 37:1–109, 1985) and have engendered a burgeoning field of research into small molecule mimetics of them (e.g., Hanessian et al., Tetrahedron 53:12789–12854, 1997). Many mimetics have either been external turn-mimetics which do not allow for the display of all the physiologically relevant side-chains (e.g., Freidinger et al., Science 210:656–8, 1980) or small, conformationally mobile cyclic peptide derivatives (e.g., Viles et al., Eur. J. Biochem. 242:352–62, 1996). However, non-peptide compounds have been developed which closely mimic the secondary structure of reverse-turns found in biologically active proteins or peptides. For example, U.S. Pat. Nos. 5,475,085, 5,670,155 and 5,672,681 to Kahn and published PCT WO94/03494 to Kahn all disclose conformationally constrained, non-peptidic compounds which mimic the three-dimensional structure of reverse-turns. More recently, U.S. Pat. No. 5,929,237 to Kahn, published PCT WO97/15577 to Kahn, published PCT WO98/49168 to Kahn et al., U.S. Pat. No. 6,013,458 to Kahn et al., U.S. Pat. No. 6,184,223 to Kahn et al. and published PCT WO01/16135A2 to Stasiak et al. disclosed additional, highly constrained bicyclic heterocycles as reverse-turn mimetics. Nevertheless, as no one template can mimic every type of turn, there remains a need in the art for additional reverse-turn templates.
Cell adhesion is critical to the viability of living organisms. Adhesion holds multicellular tissues together and directs embryonic development. It plays important roles in wound healing, eradication of infection and blood coagulation. Integrins are a family of cell surface proteins intimately involved in all of these functions. They have been found in nearly every type of human cell except red blood cells. Abnormalities in integrin function contribute to a variety of disorders including inflammatory diseases, heart attack, stroke, and cancer.
Integrins consist of heterodimers of α and β subunits, non-covalently bound to each other. These cell surface receptors extend through the cell membrane into the cytoplasm. At least 15 different α and 9 different β subunits are known. However, because most a proteins associate with only a single β there are about 21 known integrin receptors. On the cell surface the heads of the two subunits contact each other to form a binding surface for extracellular protein ligands, allowing attachment to other cells or to the extracellular matrix. The affinity of these receptors may be regulated by signals from outside or within the cell. For example, recruitment of leukocytes to the site of injury or infection involves a series of adhesive interactions. Weak interaction between endothelial and leukocyte selectins and carbohydrates mediate transient adhesion and rolling of the leukocyte along the vessel wall. Various chemokines and other trigger factors released by the site of inflammation serve as signals to activate integrins from a quiescent to a high affinity state. These activated integrins then bind their cognate ligands on the surface of the endothelial cells, resulting in strong adhesion and flattening of the leukocyte. Subsequently the leukocyte migrates through the endothelium into the tissue below.
Integrin α4β1 mediates cell adhesion primarily through binding to either vascular cell adhesion molecule-1 (VCAM-1) or an alternatively spliced variant of fibronectin containing the type III connecting segment (IIICS). A variety of cells involved in inflammation express α4β1, including lymphocytes, monocytes, basophils and eosinophils, but not neutrophils. Monoclonal antibodies to the α4 subunit have been used to validate α4-containing integrins as potential therapeutic targets in animal models of rheumatoid arthritis (Barbadillo et al., Springer Semin Immunopathol. 16:427–36, 1995; Issekutz et al., Immunology 88:569–76, 1996), acute colitis (Podolsky et al., J. Clin. Invest. 92:372–80, 1993), multiple sclerosis (Yednock et al., Nature 356:63–6, 1992), asthma (Abraham et al., J. Clin. Invest. 93:776–87, 1994); U.S. Pat. No. 5,871,734) and diabetes (Tsukamoto et al., Cell Immunol. 165:193–201, 1995). More recently, low molecular weight peptidyl derivatives have been produced as competitive inhibitors of α4β1 and one has been shown to inhibit allergic airway responses in sheep (Lin et al., J. Med. Chem. 42:920–34, 1999).
It has been shown that a key sequence in IIICS involved in binding to α4β1 is the 25 residue peptide CS1, and within that sequence the minimally recognized motif is the tripeptide, LDV. A similar sequence, IDS, has been implicated in the binding of VCAM-1 to α4β1. X-ray crystal structures of an N-terminal two-domain fragment of VCAM-1 show that the IDS sequence is part of an exposed loop linking two beta-strands (Jones et al., Nature 373:539–44, 1995; Wang et al., Proc. Natl. Acad. Sci. USA 92:5714–8, 1995). Cyclic peptides and derivatives thereof which adopt reverse-turn conformations have proven to be inhibitors of VCAM-1 binding to α4β1 (WO 96/00581; WO 96/06108; Doyle et al., Int. J. Pept. Protein Res. 47:427–36, 1996). In addition, a number of potent and selective (versus α5β1) cyclic peptide-based inhibitors have been discovered (Jackson et al., J. Med. Chem. 40:3359–68, 1997). Several non-peptidyl beta-turn mimetics have also been reported to bind α4β1 with IC50s in the low micromolar range (Souers et al., Bioorg. Med. Chem. Lett. 8:2297–302, 1998). Numerous phenylalanine and tyrosine derivatives have also been disclosed as inhibitors of α4β1 (WO 99/06390; WO 99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO 98/53817; WO 98/58902). However, no potent and orally available small molecule inhibitors have been disclosed.
A related integrin, α4β7, is expressed on the surface of lymphocytes and binds VCAM-1, fibronectin and mucosal addressin cell adhesion molecule 1 (MADCAM-1). Integrin α4β7 and MAdCAM mediate recirculation of a subset of lymphocytes between the blood, gut, and lymphoid tissue. Similar to VCAM-1 and Fibronectin CS-1 there is a tripeptide sequence, LDT, present on the CD loop of MAdCAM-1 which is important for recognition by α4β7. An X-ray crystal structure shows this sequence is also part of a turn structure (Tan et al., Structure 6:793–801, 1998). Recent studies have shown that α4β7 may also play a part in diseases such as asthma (Lobb et al., Ann. NY Acad. Sci. 796:113–23, 1996), inflammatory bowel disease (Fong et al., Immunol. Res. 16:299–311, 1997), and diabetes (Yang et al., Diabetes 46:1542–7, 1997). In addition, while α4 integrins appear to be down-regulated in carcinomas such as cervical and prostate, they appear to be up-regulated in metastatic melanoma (Sanders et al., Cancer Invest. 16:329–44, 1998), suggesting that inhibitors of α4β1 and α4β7 may be useful as anticancer agents.
Analgesia has historically been achieved in the central nervous system by opiates and analogs which are addictive, and peripherally by cyclooxygenase inhibitors that have gastric side effects. Substance P antagonists may induce analgesia both centrally and peripherally. In addition, substance P antagonists are inhibitory of neurogenic inflammation.
The neuropeptide receptors for substance P (designated as neurokinin-1) are widely distributed throughout the mammalian nervous system (especially brain and spinal ganglia), the circulatory system and peripheral tissues (especially the duodenum and jejunum) and are involved in regulating a number of diverse biological processes. Such processes include sensory perception of olfaction, vision, audition and pain, movement control, gastric motility, vasodilation, salivation, and micturition (Pernow, Pharmacol. Rev., 35:85–141, 1983). Additionally, the neurokinin-1 and neurokinin-2 receptor subtypes are implicated in synaptic transmission (Laneuville et al., Life Sci. 42:1295–1305, 1988).
The receptor for substance P is a member of the superfamily of G protein-coupled receptors. This superfamily is an extremely diverse group of receptors in terms of activating ligands and biological functions. In addition to the tachykinin receptors, this receptor superfamily includes the opsins, the adrenergic receptors, the muscarinic receptors, the dopamine receptors, the serotonin receptors, a thyroid-stimulating hormone receptor, the product of the oncogene ras, the yeast mating factor receptors, a Dictyostelium cAMP receptor, and receptors for other hormones and neurotransmitters (Hershey et al., J. Biol. Chem. 226:4366–4373, 1991).
Substance P is a naturally occurring undecapeptide belonging to the tachykinin family of peptides, the latter being so-named because of their prompt contractile action on extravascular smooth muscle tissue. The tachykinins are distinguished by a conserved carboxyl-terminal sequence Phe-X-Gly-Leu-Met-NH2. In addition to substance P the known mammalian tachykinins include neurokinin A and neurokinin B. The current nomenclature designates the receptors for substance P, neurokinin A, and neurokinin B as neurokinin-1, neurokinin-2, and neurokinin-3 respectively. More specifically, substance P is a neuropeptide that is produced in mammals and possesses a characteristic amino acid sequence (Chang et al., Nature New Biol. 232:86, 1971; Veber et al., U.S. Pat. No. 4,680,283). In mammals, substance P acts as a vasodilator, a depressant, stimulates salivation and produces increased capillary permeability. It is also capable of producing both analgesia and hyperalgesia, depending on dose and pain responsiveness of the mammal (Frederickson et al., Science 199:1359, 1978; Oehme et al., Science 208:305, 1980) and plays a role in sensory transmission and pain perception (Jessell et al., Advan. Biochem. Psychopharmacol. 28:189, 1981). For example, substance P is believed to be involved in the neurotransmission of pain sensations (Otsuka et al., “Role of Substance P as a Sensory Transmitter in Spinal Cord and sympathetic Ganglia” in 1982 Substance P in the Nervous System, Ciba Foundation Symposium, 91, 13–34 (published by Pitman); Otsuka et al., Trends Pharmacol. Sci. 8:506–510, 1987), specifically in the transmission of pain in migraine (Sandberg et al., J. Med. Chem. 25:1009, 1982; Moskowitz et al., Trends Pharmacol. Sci. 13:307–311, 1992), and in arthritis (Levin et al., Science 226:547–549, 1984); Lotz et al., Science 235:893–895, 1987). Substance P may also play a role in demyelinating diseases such as multiple sclerosis and amyotrophic lateral sclerosis (Luber-Narod et. al., poster C.I.N.P. XVIIIth Congress, 28th Jun.–2nd Jul., 1992), and in disorders of bladder function such as bladder detrusor hyperreflexia (Lancet, 16th May, 1239, 1992). Tachykinins have also been implicated in gastrointestinal (GI) disorders and diseases of the GI tract, such as inflammatory bowel disease (Mantyh et al., Neuroscience 25:817–37, 1988; Regoli in “Trends in Cluster Headache” Ed. F. Sicuteri et. al., Elsevier Scientific Publisher, Amsterdam, pp. 85–95, 1987) and emesis (Trends Pharmacol. Sci. 9:334–341, 1988; Tatersall et al., Eur. J. Pharmacol. 250, R5–R6, 1993). It is also hypothesized that there is a neurogenic mechanism for arthritis in which substance P may play a role (Kidd et al., Lancet, Nov. 11, 1989; Gronblad et al., J. Rheumatol. 15:1807–1810, 1988). Therefore, substance P is believed to be involved in the inflammatory response in diseases such as rheumatoid arthritis and osteoarthritis (O'Byrne et. al., Arthritis and Rheumatism 33:1023–1028, 1990).
Tachykinin receptor antagonists are believed to be useful for treatment of pain, headache (especially migraine), Alzheimer's disease, multiple sclerosis, attenuation of morphine withdrawal, cardiovascular changes, oedema, such as oedema caused by thermal injury, chronic inflammatory diseases such as rheumatoid arthritis, asthma/bronchial hyperreactivity and other respiratory diseases including allergic rhinitis, inflammatory diseases of the gut including ulcerative colitis and Chrohn's disease, ocular injury and ocular inflammatory diseases, proliferative vitreoretinopathy, irritable bowel syndrome and disorders of bladder function including cystitis and bladder detruser hyperreflexia (Maggi et al., J. Auton. Pharmacol. 13:23–93, 1993; Snider et al., Chem. Ind. 1:792–794, 1991). Other disease areas where tachykinin antagonists are believed to be useful are, allergic conditions (Hamelet et al., Can. J. Pharmacol. Physiol. 66:1361–1367, 1988), immunoregulation (Lotz et al., Science 241:1218–1221, 1988; Kimball et al., J. Immunol. 141:3564–3569, 1988; Perianin et. al., Biochem. Biophys. Res. Commun. 161:520, 1989), postoperative pain and nausea (Bountra et al., Eur. J. Pharmacol. 249:R3–R4, 1993; Tattersall et. al., Neuropharmacology 3:259–260, 1994), vasodilation, bronchospasm, reflex or neuronal control of the viscera (Mantyh et. al., Proc. Natl. Acad. Sci. USA 85:3235–3239, 1988) and, possibly by arresting of slowing β-amyloid-mediated neurodegenerative changes (Yankner et al., Science 250:279–282, 1990) in senile dementia of the Alzheimer type, Alzheimer's disease and Downs Syndrome. Tachykinin antagonists may also be useful in the treatment of small cell carcinomas, in particular small cell lung cancer (SCLC) (Langdon et al., Cancer Research 52:4554–4557, 1992). It has further believed that tachykinin receptor antagonists have utility in the following disorders: depression, dysthymic disorders, chronic obstructive airways disease, hypersensitivity disorders such as poison ivy, vasospastic diseases such as angina and Reynauld's disease, fibrosing collagen diseases such as scleroderma and eosinophillic fascioliasis, reflex sympathetic dystrophy such as shoulder/hand syndrome, addiction disorders such as alcoholism, stress related somatic disorders, neuropathy, neuralgia, disorder related to immune enhancement of suppression such as systemic lupus erythmatosus (EPO Pub. No. 0,436,334), ophthalmic diseases such as conjunctivitis, vernal conjunctivitis, and the like, and cutaneous diseases such as contact dennatitis, atopic dermatitis, urticaria, and other eczematoid dermatitis (EPO Pub. No. 0,394,989).
Substance P receptor antagonists may be useful in mediating neurogenic mucus secretion in mammalian airways and hence provide treatment and symptomatic relief in diseases characterized by mucus secretion, in particular, cystic fibrosis (Ramnarine et al., abstract presented at 1993 ALA/ATS Int'l Conference, 16–19 May, 1993, published in Am. Rev. of Respiratory Dis., May 1993). Neurokinin-1 receptor antagonists alone or in combination with bradykinin receptor antagonists may also be useful in the prevention and treatment of inflammatory conditions in the lower urinary tract, especially cystitis (Giuliani et al., J. Urology 150:1014–1017, 1993). Furthermore, antagonists selective for the neurokinin-1 and/or neurokinin-2 receptor may be useful in the treatment of asthmatic disease (Frossard et. al., Life Sci. 49:1941–1953, 1991; Advenier et al., Biochem. Biophys. Res. Comm. 184:1418–1424, 1992; Barnes et al., Trends Pharmacol. Sci. 11:185–189, 1993).
The following documents relate to compounds that exhibit activity as neurokinin antagonists: U.S. Pat. No. 6,194,406 B1; U.S. Pat. No. 6,191,135 B1; U.S. Pat. No. 6,177,450 B1; U.S. Pat. No. 6,147,083; U.S. Pat. No. 6,110,919; U.S. Pat. No. 6,063,926; U.S. Pat. No. 6,048,859.
While significant advances have been made in the synthesis and identification of conformationally constrained, reverse-turn mimetics, there is still a need in the art for small molecules that mimic the secondary structure of peptides. There is also a need in the art for libraries containing such members, particularly those small templates capable of supporting a high diversity of substituents. In addition, there is a need in the art for techniques for synthesizing these libraries and screening the library members against biological targets to identify bioactive library members. Further, there is a need in the art for small, orally available inhibitors of integrins, for use in treating inflammatory diseases and cardiovascular diseases, as well as some cancers. In particular there is a need for inhibitors of α4β1 and α4β7, for use in the treatment of rheumatoid arthritis, asthma, diabetes and inflammatory bowel disease. Further, there is a need in the art for small, orally available inhibitors of neurokinins, for use in treating inflammatory diseases, central nervous system disorders, as well as several other disorders. In particular there is a need for inhibitors of neurokinin-1, neurokinin-2, and neurokinin-3, for use in the treatment or prevention of various mammalian disease states, for example asthma, cough, chronic obstructive pulmonary disease (COPD), bronchospasm, emesis, neurodegenerative disease, ocular disease, inflammatory diseases such as arthritis, central nervous system conditions such as migraine and epilepsy, nociception, psychosis, and various gastrointestinal disorders such as Crohn's disease.
The present invention fulfills these needs and provides further related advantages.